PROJECT SUMMARY Chromatin decondensation is controlled in part by the PAD4-dependent conversion of arginine to citrulline on histones (i.e. histone citrullination). PAD4 is highly expressed in neutrophils and is the only member of the peptidylarginine deiminase (PAD) family expressed in the nucleus. Hypercitrullination by PAD4 is pathophysiological and associated with many diseases (e.g. autoimmune disease, inflammation, and cancer). PAD4 activation in neutrophils initiates leukotoxic hypercitrullination (LTH), a cell death program distinct from apoptosis, necrosis, or traditional NETosis, wherein chromatin is hypercitrullinated, decondensed, and extruded from the cell in NET-like structures. Of note, LTH NETs (neutrophil extracellular traps) are distinct from NADPH oxidase-dependent NETs (i.e. traditional NETosis; an antibiotic form of neutrophil cell death), which do not contain citrullinated histones. The release of citrullinated chromatin into the blood is thought to contribute directly to PAD4-related pathologies. Thus, PAD4 is a valuable drug target and nucleosome citrullination is a promising blood-accessible biomarker. There are no quantitative assays capable of reliably quantifying PAD4-dependent nucleosome citrullination in human neutrophils or blood. Current PAD4 ELISAs are low-throughput and use citrullinated histones (CitHis) as calibration standards. CitHis are problematic for quantification for two major reasons. First, they fail to provide accurate quantification of nucleosomes at physiological levels observed in cells; second, free histones (not assembled into a nucleosome) readily aggregate in plasma, thereby limiting their use in blood- based samples. Here, we propose to develop the first assay (CitNucTM) to accurately quantify histone citrullination for next-generation drug discovery and biomarker development. Our innovative assay uses chemically-defined recombinant citrullinated designer nucleosomes (CitH3-dNucs) as quantification standards for reliable assay calibration. Unlike histones, citrullinated nucleosomes provide useful standards at low concentration and do not aggregate in heterogeneous biological solutions (i.e. plasma). Moreover, since CitH3- dNucs resemble chromatin in vivo, samples require minimal processing enabling development of the first high- throughput (HT) cell-based assays for PAD4 inhibitor development. In Aim 1, we will develop the CitNuc assay (on AlphaLISA platform). This high-throughput-compatible assay will be useful for drug development and basic research applications. In Aim 2, we will develop the pre-clinical application of our CitNuc assay to quantify citrullinated nucleosomes levels in plasma. To validate this assay, we will compare citrullinated nucleosome levels in patients with rheumatoid artheritis (RA), a disease characterized by high levels of citrullinated nucleosomes in blood. In Phase II, we will scale-up commercial manufacturing of CitH3-dNucs, develop novel methods for increased assay throughput for inhibitor screening, and expand our pre-clinical studies to develop the utility of our assay for disease monitoring in RA patients. !